The invention involves a method for measuring at least one state variable of a biological cell located in a nutrient medium, the cell being supported on and adhering to a support area, wherein at least one opening is made in the membrane of the cell for measuring the state variable. The invention further involves an apparatus for measuring at least one state variable of at least one biological cell located in a nutrient medium, the apparatus including an object carrier (specimen slide) having a support area, on which the cell can be supported in an adherent manner, and at least one measuring probe that can be brought into contact with the cell liquid located inside the cell for measuring the state variable, wherein the measuring probe is connected or combined with a measurement amplifier.
From the book, Human Physiology, Schmidt; Thews (publisher), 23rd edition (1987), pages 20-21, an apparatus of this type is already known, which has a suction device connected to a hollow needle with an inner cavity that has an opening on the open end of the hollow needle. In the inner cavity of the hollow needle, a measuring probe is arranged for measuring the cell potential of the cell. With this device, operating according to the so-called patch-clamp process, the hollow needle is applied on the outside of the cell membrane in order to bring the measuring electrode into contact with the cell liquid by the opening located on the free end of the hollow needle, in order to then generate a partial vacuum in the inner cavity of the hollow needle using the suction device. With this partial vacuum a piece of the cell membrane, located in front of the opening of the hollow needle, is torn out of the membrane structure. Via the resulting opening in the cell membrane, the ions located in the cell liquid get into an electrolyte located in the hollow needle and from there to the measuring probe. A reference electrode functions for determining a reference potential.
The previously known method and associated apparatus have the disadvantage that, for positioning of the hollow needle on the cell, a micromanipulator is necessary. This results in a comparatively complicated and expensive device. Moreover, the accessibility of the cells located on the specimen slide is greatly restricted by the micromanipulator. The process and apparatus are thus suitable only for an investigation of individual, or at most for a simultaneous investigation of a small number of, cells located on the specimen slide.
From U.S. Pat. No. 4,461,304 an apparatus is further known, which has a needle-shaped tip for making an opening in a cell membrane. On the tip a plurality of sensors is arranged for neurophysiological investigations. Even with this device, for positioning of the tip on the cell, a micromanipulator is necessary.
From published European and German patent applications EP 0 689 051 A2; DE 197 12 309 A1; and EP 0 585 933 A2 and German patent DE 195 29 371 C2, apparatus are already known for measuring a cell potential, which have a specimen slide having a plurality of microelectrodes arranged in matrix form, which can be brought into connection with the outside of the membrane of a cell to be investigated. These devices make possible, however, only an extracellular measurement of the cell potential, since no opening is made in the cell membrane.
From published German patent applications DE 195 36 389 A1 and DE 195 36 384 A1 methods are already known for measuring a state variable, in which a biological component is contacted. Also with this method, an opening is not made in the biological component.
In German published patent application DE 38 16 458 A1 a microelectrode is further described, which can be used for a potentiometric or amperometric measurement in the biochemical and medical fields.
German patent DE 44 22 049 C2 discloses an ultra-microelectrode array for chemical and biochemical analyses, which has several pyramids or cone-shaped electrode tips on a substrate. According to statements of the patent document, the ultra-microelectrode array can be inserted into electrode structures for the measurement of oxygen according to Clark.
From U.S. Pat. No. 5,173,158, moreover, a process outside of the generic concept is known for generating new cells, in which cells of a first type located in a liquid are supported by a partial vacuum or hydrostatic pressure on a support area of a porous layer, such that the cells engage with a component in the pores of the porous layer. The porous layer with the cells is arranged between electrode plates bordering on the liquid to which an electric voltage is applied, which opens the cell membrane of the cells in the component that engages with the pores. Thereafter, cells of a second type are brought into the insides of the cells of the first type through these openings.
Thus, an object of the present invention is to create a method and apparatus of the type mentioned at the outset, which makes possible a simple measurement of a state variable of the cells. In particular, a costly manual positioning of a hollow needle on the cells to be investigated should be avoided.
This object is achieved with respect to the method in that the opening in the cell membrane is made within the support area of the cell and spaced from the support edge, and through this opening the state variable is measured.
In this way it is possible to arrange a poration agent or a poration tool, which is used for making the cell membrane opening, in the support area of the cells on the specimen slide so that the cell is also positioned simultaneously on the poration agent or poration tool when it is supported on the support area. In this way, a costly manual positioning of a poration tool can be omitted. Since the opening is formed in the cell membrane within the support area of the cells and at a distance from the edge of the support area, the membrane area of the cells, which surrounds the opening and is adheringly attached to the support area, seals off the opening from the nutrient liquid. The cell liquid located inside the cell is thus electrically insulated to the greatest extent possible from nutrient liquid. The measured state variable of the cells can, for example, be an ion concentration, a gas quantity, a temperature or any other desired physical, chemical or biological characteristic of a cell.
In order to measure the cell potential of the cells, the electrical voltage between the cell liquid and the nutrient medium can be measured through the opening formed in the cell membrane. Using this process, for example, electrical signals transmitted between nerve cells can be tested. Using this process, electric d.c. and/or a.c. voltage potentials, especially potentials that change quickly in time, can be measured.
In an especially advantageous embodiment of the invention, the opening can be made using electroporation of the cell membrane. For the performance of the process, for example, an electroporation-electrode can be arranged in the support area for the cell, on which the cell is supported in an adherent manner. In order to form the opening in the cell membrane, an electric voltage then only needs to be applied between the electroporation electrode and the nutrient medium, which causes an electrical current flow that opens the cell membrane. After switching off the electroporation voltage, the electric potential can be measured through the opening of the cell membrane between the cell liquid and the nutrient medium. For this purpose, optionally, the electrode used for electroporation can also be used to measure the cell potential, so that the electrode performs a double function.
In another embodiment of the invention, at least one mechanical impulse is applied in order to form the opening in the cell membrane on a portion of the cell membrane. In this process, this portion of the cell membrane detaches from the membrane structure. Optionally , an impulse sequence can also be applied with several individual impulses.
It is especially advantageous when the opening is made in the cell membrane using ultrasound. In this process, it is even possible that the ultrasound is focused on the area of the cell membrane to be opened and/or that several ultrasonic waves are superimposed so that their oscillations are superimposed in the area of the cell membrane to be opened, to produce an oscillation having an increased amplitude. The cell membrane can be opened in this way without contact.
A contact-free opening of the cell membrane can, however, also occur in a manner such that a portion of the cell membrane is irradiated with a high-energy radiation, especially with laser radiation. In this process, the wavelength of the radiation is preferably selected such that the cell membrane absorbs the radiation well. Functionally, the radiation is coupled into the cell in the support area of the cell. Optionally, a laser beam can, however, also be coupled into the cell outside the support area, where in a membrane area located there a small coupling opening is first made, through which the laser beam is then projected through the inside of the cell onto a membrane area located in the support area of the cell, in order to cut out a portion of the membrane from the membrane structure by sweeping of the laser beam around the coupling opening.
In another embodiment of the process, the opening is made by the action of a chemical substance in the cell membrane. As a poration agent, for example, a Perforin or Triton(copyright) can be used.
It is especially advantageous if an electric and/or chemical substance and/or a substance that can be activated by radiation is used and if this substance is activated in order to make the opening in the cell membrane by the action of radiation and/or an electric field. The substance is thus activated by the supply of energy. In this process, for example, free radicals are generated, which destroy the portion of the cell membrane that is to be opened. In the inactive condition, the substance acts, for the most part, neutrally relative the cell, so that it has practically no influence on the support of the cell on the support area. A chemical substance can also be used which is chemically activated by the addition of another substance.
Another embodiment of the process provides that a portion of the cell membrane to be opened is disengaged from the membrane structure by impingement with a partial pressure and/or an excess pressure. In this process, for example, in a specimen slide that has the support area, a small opening can be provided within the support area, through which the cell is suctioned to such an extent that the membrane area located in front of the opening is torn out of the membrane structure. In order to open the cell membrane, action can be made through the opening, but also, however, an excess pressure pulse can be exerted on a membrane area of the cell.
It is advantageous that the cell be fixed on the support area by a suction force. In this way, the adhering of the cell on the support area of a specimen slide is improved. The suction force is measured selected such that the cell membrane is not mechanically damaged by the suction force.
It is advantageous when, after making the opening in the cell membrane, cell liquid is removed from the cell through the opening and examined. In this way, it is possible to detect additional cell quantities that are only measured with difficulty inside the cell. Thus, additional information about the physiology of the cell can be obtained.
It is especially advantageous when, after making the opening in the cell membrane, an intracellular manipulation can be performed through the opening, in particular, a medicine and/or a foreign substance and/or a biological substance is brought into the interior of the cell. The effects of the intracellular manipulation can then be observed by measuring a state variable of the cell. Optionally, the intracellular manipulation can also be performed, however, without a state variable of the cell being measured through the opening.
In regards to the apparatus, the previously mentioned purpose is achieved in that the measuring probe and an electric insulator surrounding it are arranged within the support area, such that the cell can be supported on the insulator to seal it from the nutrient medium, and that to open the membrane of the cell, at least one poration tool is arranged in the support area in the area of the measuring probe.
Since the poration tool is arranged within the support area, the cell can automatically be supported on the poration tool located in the support area. In an advantageous way, a costly manual positioning of the poration tool on the cell can thus be omitted. Also, auxiliary devices, such as micromanipulators, are not necessary. In this way it is possible to measure cell quantities at the same time on several cells that are arranged in close proximity to one another. After making the opening in the cell membrane, the edge of the cell membrane surrounding the opening stays in contact with the electric insulator and seals off the opening from the nutrient medium. In this way, the cell liquid located inside the cell is electrically well-insulated from the nutrient medium, so that the cell quantity can be measured through the opening of the cell membrane.
It is advantageous when the poration tool is arranged substantially concentrically around the measuring probe. The measuring probe can then come into good contact with the cell liquid located inside of the after the opening is made in the cell membrane.
In an embodiment which is provided for measuring the potential of the cell, the measuring probe is a measuring electrode, to which is allocated at least one reference electrode that can be brought into contact with the nutrient medium. With a device of this type, the difference in potential between the cell liquid and the nutrient medium can be measured in a simple way.
It is especially advantageous when the measurement electrode also is an electroporation electrode which can be connected to an electric voltage source for the electroporation of cell membrane, and when the electroporation electrode has at least one active electrode area that is arranged within the support area and which is surrounded by the electric insulator. The measuring electrode is thus simultaneously also the poration tool, and fulfills a double function, such that a device results that is constructed in an especially simply manner. The electrode connected to the measurement amplifier can, for example, be applied briefly at the potential of the voltage source using an electronic switch. Optionally, a change-over switch can also be provided, by which the measuring electrode can be selectively connected one after the other to the measurement amplifier or the voltage source. The measuring and electroporation electrode is arranged within the support area of the specimen slide so that a cell supported so as to adhere to the insulator can also optionally be supported on the active electrode area of the electrode or at least approach it until reaching the active area of an electric field that extends from the electrode. When the electroporation voltage is applied to the electrode, an electric current flows which makes an opening in the cell membrane. In the process, the edge of the cell membrane surrounding the opening stays in contact with the electric insulator and seals off the opening from the nutrient medium. The insulator resistance is independent of the cell type and is preferably larger than 10 megaohms. An offset of the potential between the nutrient medium and the cell liquid is thereby for the most part stopped. After the creation of the opening in the cell membrane, the measuring electrode is separated from the electroporation voltage source, so that then the cell potential is applied on the measuring electrode that is in contact with the cell liquid and can be measured with the measurement amplifier. The device has a simple construction and makes possible, for the most part, an automated cell potential measurement.
In an advantageous embodiment of the invention it is provided that the poration tool is an electroporation electrode that is set off at a distance from the measuring probe. The electrode can be connected to an electric voltage source for the electroporation of the cell membrane. The electroporation electrode has at least one active electrode area arranged within the support area and surrounded by the electric insulator. The electroporation electrode is thus separated from the measuring probe. In this way, the effect of the capacitance of the supply lines from the voltage source to the electroporation electrode is reduced to the measurement signal determined by the measuring probe. Thus, changes of the cell potential that are rapid in time, as occurs in nerve cells for example, can be measured even better.
In an especially advantageous further embodiment of the invention, it is provided that the active electrode area of the electroporation electrode has at least one sharp tip or edge and is preferably arranged to project beyond the surface plane of the support area. The active electrode area can thus contact the cell membrane of a cell supported on the support area on the outside. In this way, a good electrical contact between the electrode and the cell membrane is possible. The projecting active electrode area additionally allows a good electrical contact with the cell liquid located inside the cell through the opening of the cell membrane and, above and beyond that, acts to oppose a closing, through cell repair mechanisms, of the opening made by electroporosis in the cell membrane.
It is advantageous if the electroporation electrode is constructed as a hollow electrode which has an inner cavity with an opening located on the surface of the support area, and if the measuring electrode is a rod electrode arranged in the inner cavity of the electroporation electrode, the free end of the rod electrode preferably extending up to the opening of the inner cavity. Inside the hollow electrode, an electrolyte can be arranged which can correspond, for example, to the nutrient medium, so that after the opening of the cell wall, charged particles, especially ions, contained in the cell liquid can migrate through this electrolyte to the electrode. Thus, a larger electrode surface can come into contact with the charged particles. The electric contact between the measuring electrode and the cell liquid is improved by this.
It is especially advantageous when immediately adjacent to the electroporation-electrode a switching component, in particular a static switch, is arranged with which the electroporation electrode can be connected to the electroporation voltage source. The connection line between the electroporation electrode and the electroporation voltage source can then be uninterrupted closely adjacent to the electrode, so that the parasitic capacitance of this connection line is uncoupled from the measuring probe during the measurement of the cell potential. In this way, cell potential voltages that change quickly in time can be measured more exactly. Preferably, the static switch is a low-noise junction-field effect-transistor.
In an advantageous embodiment of the apparatus, the poration tool for opening the cell membrane can be moved using at least one actuator, in particular a piezo element, across the surface of the support area relative to the specimen slide. With this device, the opening is thus made mechanically in the cell membrane. In the process, the poration tool can even be alternately moved away from and toward the cell membrane. For this purpose, the actuator can be connected with a drive device for generating an ultrasonic oscillation. The poration tool can be alternately moved relative to the specimen slide in a direction that runs perpendicular or in a direction that is oblique to the surface of the support area.
It is advantageous when the poration tool has at least one sharp tip or edge that can be brought into contact with the membrane of the cell. The cell membrane can then be better opened by moving the poration tool.
It is especially advantageous when the measuring probe is at the same time also the poration tool and for this can be moved, using the actuator or piezo element, across the surface of the support area relative to the specimen slide. The measuring probe then fulfills a double function, such that an additional poration tool can be omitted.
In another advantageous embodiment, the specimen slide has an optical window in the area of the measuring probe, which is arranged in the beam path of a laser beam in order to open the cell membrane. Using this device, a portion of the cell membrane can be irradiated briefly with high-energy optical radiation, where this area is heated to such an extent, that an opening is made in the cell membrane.
It is especially advantageous when, in order to generate the laser beam, a laser diode is integrated into the specimen slide. In this process, the laser diode can even be arranged directly behind the opening, so that the laser beam can be coupled directly and thus, for the most part, free of losses into the cell membrane supported on the support area.
Functionally, the measuring probe is arranged substantially concentrically around the optical window. The measuring probe can then come into better contact with the cell liquid after opening the cell membrane.
In an advantageous embodiment is provided that the poration tool has, in order to open the cell membrane, a chemical substance and/or at least one outlet opening connected to the supply channel for a chemical substance. The opening can thus also be made in the cell membrane chemically, where as a poration agent, for example, Perforin or Triton(copyright) can be used.
In another embodiment the poration tool has at least one channel that opens into the support area, by the use of which a portion of the cell membrane can be impinged in order to make the opening in the cell membrane with a partial vacuum and/or excess pressure. In a device of this type, the opening is thus made in the cell membrane using a partial vacuum or excess pressure, where the partial vacuum or excess pressure is switched off after the opening of the cell membrane. During an opening of the cell membrane by partial vacuum, a suction of the cell liquid out of the inside of the cell is prevented to the greatest extent possible. Correspondingly, during opening of the cell membrane using excess pressure, it is prevented that a medium located in the channel, that is preferably a fluid, can enter into the inside of the cell. In order to switch off the partial vacuum or excess pressure, for example a pressure change occurring during the opening of the cell membrane in the channel can be determined. The partial vacuum or excess pressure can be generated using a suitable auxiliary device, for example a pump, or hydrostatically. In an advantageous way, the channel can also be used prior to supporting the cell, in order to suction off nutrient medium from the support area, so that a current occurs in the nutrient medium, which conducts the cells located therein to the opening of the channel arranged in the area of the measurement electrode.
It is especially advantageous when the measuring probe is constructed as a hollow sensor that is installed into the surface of the specimen slide, and has at least one inner cavity, wherein the inner cavity has an opening on the surface of the support area. In the inside of the hollow electrode, an electrolyte can be arranged, so that charged particles contained in the cell liquid can migrate through the electrolyte to the measuring probe. Thus, a larger surface of the measuring probe can come contact with the charged particles, which improves the electrical contact between the measuring electrode and the cell liquid.
An advantageous embodiment provides that the electric insulator within the support area has a projection that projects beyond the surface plane of the support area, and that the measuring probe is arranged on the free end of the projection, which faces away from the surface of the support area. In this way, a good electrical and/or mechanical contact results between the measuring probe and the cell membrane.
Functionally, it is provided that the cross-section of the projection proceeding out from the surface plane of the support area tapers to the point that projects furthest out. The cell then adheres better with its membrane to the projection of the electric insulator. In addition, the insulator can, in production engineering in the manufacture of the specimen slide, be better applied as a coating on the area of the projection that tapers.
In an advantageous further embodiment of the invention, it is provided that the specimen slide has a profile in the support area, which has at least one profile recess and/or a profile projection that surrounds the measuring probe. In this way, a better seal of the cell liquid is achieved against the nutrient medium by the cell membrane that adheres to the insulator.
It is advantageous when the profile recess and/or profile projection is interrupted in the extension direction by at least one gap. The cell can then better adhere to the surface of the specimen slide in the area of the profile. The profile projection or profile recess can, for example, have a web structure or a structure in the manner of a checkerboard pattern.
It is especially advantageous if the profile recess and/or the profile projection is constructed in a ring-shape, and if preferably several such ring-shaped profile recesses and/or profile projections are arranged essentially concentrically to the measuring probe. Thus, radially to the measuring probe, several profile recesses and/or projections are connected behind each other or interlaced within each other, so that the cell liquid is even better sealed off from the nutrient medium.
A preferred embodiment of the invention provides that the insulator is an insulation layer arranged on the surface of the profile. In an advantageous manner, the path for a leak current flowing on the surface of the insulator from the cell liquid to the nutrient medium is enlarged by the insulation layer profiled in this way, so that the measuring probe or the measuring electrode is insulated even better from the nutrient medium.
Another embodiment provides that the profile projection(s) is (are) mounted on the surface of the insulator. The specimen slide can then be manufactured in a more simple way by production engineering.
It is especially advantageous when in the support area of the specimen slide on its surface, at least one coating that has a cell adhesion protein and/or a hydrophilic coating is arranged. The cell membrane then adheres better to the specimen slide. The cell adhesion coating can for example have laminin, fibronectin, or poly-L-lysine. Possibly, a hydrophobic coating can also be arranged on the edge of the support area that borders the electrode, with bonding positions for the hydrophobic lipids located in the cell membrane.
It is advantageous when as a mechanical guide for the cells, boundary walls are arranged on both sides of the measuring probe, which preferably delimit a groove-like guide channel. In this manner, the measuring electrode(s) is (are) preferably arranged in the middle between the boundary walls at the base of the groove of the guide channel, so that cells located in the guide channel can move essentially only in the extension direction of the guide channel and then inevitably come into contact with the measuring probe.
In an especially advantageous further embodiment of the invention, a field-effect transistor (FET), in particular a junction-field-effect transistor (J-FET) is arranged adjacent to the measuring probe, and the measuring probe is connected for the impedance transformation of the measuring signal to the gate of the FET. In the process, the coupling of the measuring electrode onto the gate is done capacitively in a metal oxide-field effect transistor (MOS-FET), wherein the measuring electrode is preferably arranged directly above the gate of the MOS-FET that is installed into the surface of the specimen slide. In an advantageous way, a junction-FET makes possible a high-ohmic, but nevertheless, low-noise coupling of an intracellular electrical signal. The low input capacitance of the junction-FET allows, in particular, even for rapid changes in the cell potential, obtaining a measuring signal that is, for the most part, free from feedback. By the impedance conversion directly at the measuring site, the screening cost for the connection lines from the measuring electrode to a measurement amplifier and/or an evaluation device can be reduced. The field effect transistors manufactured by the manufacturing processes known in semiconductor technology allow, beyond that, a high integration density.
It is advantageous when at least one liquid channel opens in the support area of the specimen slide, adjacent to the measuring probe, preferably in an area surrounded by this. Through this liquid channel, after the opening of the cell membrane, a small quantity of cell liquid can be suctioned off and/or a biological substance, for example a gene, and/or a foreign substance, a medication or the like, whose effect on the cell is to be investigated, can be added to the cell liquid. To add a substance, the device can optionally also be used without a measuring probe.
Expediently, in the course of the liquid channel, a micropump preferably integrated in the specimen slide is arranged. Optionally, several micropumps can even be allocated to one liquid channel which, for example, can respectively be connected to a cavity located in the specimen slide to support a liquid or a medium that is to be cytologically tested.
It is especially advantageous when within the liquid channel, preferably in a wall of the liquid channel, at least one microsensor is arranged for measuring a quantity of the cell. In this way, additional intracellular parameters, such as ion concentrations, gas contents, enzyme and/or protein concentrations, can be determined.
It is advantageous when, in order to generate an electric field that conducts the cell to the measuring probe, at least one additional electrode is arranged in the support area and/or adjacent to it. An electric field can thereby be generated on the surface of the specimen slide, which exerts a force on the biological cells, whose dielectric constants are distinguished from those of the nutrient medium in which they are arranged. This force guides the cells to the measuring probe.
An especially advantageous further embodiment of the invention provides that in the support area several measuring probes and electroporation electrodes are preferably arranged as an array, and that to each of these measuring probes at least one poration tool is allocated. Such a device allows a locationally triggered measurement of the cell potential on a cell population. In this process, the cell potential can be measured on many cells that are close to each other at the same time. In this way, it is possible, for example, to perform quasi static cell membrane potential measurements on nerve or tumor cells, in order to monitor their electric activity. In this way, it is even possible to monitor the information transfer between the cells, through the locationally triggered and time-triggered measurement of the cell potential, in a cell composite with nerve cells connected through synapses. Optionally, a multiplexer can also be integrated into the specimen slide, by which several measurement and/or electroporation electrodes can be connected alternatingly one after the other with a measurement signal guide, such that the number of the supply lines to the specimen slide constructed as a sensor chip is correspondingly reduced.